Difference between revisions of "Part:BBa K3924055"
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K3924055 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3924055 SequenceAndFeatures</partinfo> | ||
| − | + | ==Usage and Biology== | |
| + | In order to heal the intestinal tract damage, one of notable symptoms of IBD, we adopted a special therapy expressing the therapeutic proteins controllably by <i>E.coli Nissle 1917</i> (EcN) in situ. The design is based on a ternary system: sensor - secretion peptide - therapeutic proteins.<br/> | ||
| + | [[Image:T--Tsinghua--General design of the treatment ternary system.png|center|600px|thumb|'''Figure 1: General design of the treatment ternary system''']] | ||
| + | DsbA is one of candidate secretion peptides we screened out, which is a most essential element that help our therapeutic protein secrete outside the engineered bacteria and diffuse inside the patient's intestinal tract. It is a siganl peptide of <i>Escherichia coli</i> thiol:disulfide oxidoreductase DsbA<sup>[1]</sup>. The sequence is mainly based on literature we had reviewed and modified by our condon preference system.<br/> | ||
| + | ==Functional Verification== | ||
| + | For all candidate secretion peptides, we did codon analysis with our own software tool.(Figure 2) | ||
| + | [[Image:T--Tsinghua--Codon preference confidence analysis for secretion peptide.png|center|600px|thumb|'''Figure 2.Codon preference confidence analysis for secretion peptide, in theroy, the total GC% of EcN is 49.13%, 1st letter GC% is 55.38%, 2nd letter GC% is 42.34%, and 3rd letter GC% is 50.58%. We compare P2N and GenScript® online codon preference tool (GenSmart) analysis results for the bias from theoretical values. The lighter the squares are, the better for the codon optimization. (DNA sequence of each protein is detailed in the part page) | ||
| + | ''']] | ||
| + | As for DsbA, the result of codon preference is shown in Figure 3. | ||
| + | [[Image:T--Tsinghua--Codon preference confident analysis of DsbA.png|center|600px|thumb|'''Figure 3.Codon preference confident analysis of DsbA''']] | ||
| + | The workflow of the verification of the secretion peptides' function is shown in Figure 4 | ||
| + | [[Image:T--Tsinghua--Secretion peptide flowchart.png|center|600px|thumb|'''Figure 4: Secretion peptide flowchart''']] | ||
| + | The functional verification of secretion peptides was conducted by checking the fluorescence of the bacteria supernatant after centrifuging at 8000 rpm for 1 minute. The fluorescence is measured by microplate reader. The results are shown in Figure 5.<br/> | ||
| + | [[Image:T--Tsinghua--Fluorescence intensity.png|center|600px|thumb|'''Figure 5: Fluorescence intensity''']] | ||
| + | With RGP-GFP group (RGP is the plasmid backbone in our design) as a negative control, which doesn’t have any secretion peptide to diffuse GFP out of the protein, RGP-DsbA-GFP, however, does not show a significant difference. The fluorescence is slightly higher, but maybe due to the volatile lab environment, the significance cannot be shown. Nevertheless, we evaluate this part as a success.<br/> | ||
| + | ==Reference== | ||
| + | [1] Zhou Y Z, Liu P, Gan Y T, et al.Enhancing full-length antibody production by signal peptide engineering.Microbial Cell Factories, 2016,15(1):1-11.<br/> | ||
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Latest revision as of 20:57, 21 October 2021
DsbA-GFP
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
In order to heal the intestinal tract damage, one of notable symptoms of IBD, we adopted a special therapy expressing the therapeutic proteins controllably by E.coli Nissle 1917 (EcN) in situ. The design is based on a ternary system: sensor - secretion peptide - therapeutic proteins.
DsbA is one of candidate secretion peptides we screened out, which is a most essential element that help our therapeutic protein secrete outside the engineered bacteria and diffuse inside the patient's intestinal tract. It is a siganl peptide of Escherichia coli thiol:disulfide oxidoreductase DsbA[1]. The sequence is mainly based on literature we had reviewed and modified by our condon preference system.
Functional Verification
For all candidate secretion peptides, we did codon analysis with our own software tool.(Figure 2)
As for DsbA, the result of codon preference is shown in Figure 3.
The workflow of the verification of the secretion peptides' function is shown in Figure 4
The functional verification of secretion peptides was conducted by checking the fluorescence of the bacteria supernatant after centrifuging at 8000 rpm for 1 minute. The fluorescence is measured by microplate reader. The results are shown in Figure 5.
With RGP-GFP group (RGP is the plasmid backbone in our design) as a negative control, which doesn’t have any secretion peptide to diffuse GFP out of the protein, RGP-DsbA-GFP, however, does not show a significant difference. The fluorescence is slightly higher, but maybe due to the volatile lab environment, the significance cannot be shown. Nevertheless, we evaluate this part as a success.
Reference
[1] Zhou Y Z, Liu P, Gan Y T, et al.Enhancing full-length antibody production by signal peptide engineering.Microbial Cell Factories, 2016,15(1):1-11.